Interaction of a new fluorescent ATP analogue with skeletal muscle myosin subfragment-1

A new fluorescent ribose-modified ATP analogue, 2'(3')-O-{6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic}-ATP (NBD-ATP), was synthesized and its interaction with skeletal muscle myosin subfragment-1 (S-1) was studied. NBD-ATP was hydrolysed by SI at a rate and with divalent cation-dependence similar to those in the case of regular ATP. Skeletal HMM supported actin translocation using NBD-ATP and the velocity was slightly higher than that in the case of regular ATP. The addition of S1 to NBD-ATP resulted in quenching of NBD fluorescence. Recovery of the fluorescence intensity was noted after complete hydrolysis of NBD-ATP to NBD-ADP. The quenching of NBD-ATP fluorescence was accompanied by enhancement of intrinsic tryptophan fluorescence. These results suggested that the quenching of NBD-ATP fluorescence reflected the formation of transient states of ATPase. The formation of S-1.NBD-ADP-BeFn. and S-1.NBD-ADP.AlF4- complexes was monitored by following changes in NBD fluorescence. The time-course of the formation fitted an exponential profile yielding rate constants of 7.38 x 10(-2) s(-1) for BeFn and 1.1 x 10(-3) s(-1) for AlF4-. These values were similar to those estimated from the intrinsic fluorescence enhancement of trp due to the formation of S1 1.ADP.BeFn or AlF4- reported previously by our group. Our novel ATP analogue seems to be applicable to kinetic studies on myosin.